Calcium/calmodulin-dependent protein kinase II (CaM kinase II) is ubiquitously expressed and involved in many different signal transduction pathways. In addition to phosphorylating a wide array of substrates, the kinase can phosphorylate itself to achieve a calcium/calmodulin-independent state. The mechanism of autophosphorylation, which requires two calmodulin molecules to both activate the kinase and present another subunit for phosphorylation, suggests that CaM kinase II may serve as a calcium spike frequency decoder, a function that may be critical in such frequency-dependent phenomena as synaptic long-term potentiation and control of cardiac contraction. Quantitative mechanistic study of kinase autoregulation requires precise control of kinase exposure to experimental solutions, which is achieved with a lab-built multi-valve flow system and immobilized kinase. This device will be used to study the kinetics and thermodynamics of autoregulation, and the effects of various small molecules, as well as kinase and calmodulin mutation, on this process.